The present invention relates to the field of recombinant DNA technology, more in particular to the field of gene therapy. Specifically, the present invention relates to gene therapy using materials derived from adenovirus, in particular human recombinant adenovirus, and relates to novel virus derived vectors and novel packaging cell lines for vectors based on adenoviruses. Furthermore, this invention also pertains to the screening of replication-competent and revertant E1 and/or E2A adenoviruses from recombinant adenoviruses used in gene therapy.
The current generation of adenoviral vectors for gene therapy contains deletions of the early region 1 (xe2x80x9cE1xe2x80x9d), where new genetic information can be introduced. The E1 deletion renders the recombinant virus replication defective. It was generally thought that E1-deleted vectors would not express any other adenoviral genes, because E1 is reported to trigger the transcription of the other adenoviral genes. It has been shown by us and others that these vectors express several early (e.g., E2A) and late genes (e.g., fiber and penton-base) in the absence of E1. This means that delivery of a therapeutic gene using E1-deleted adenoviral vectors will result in expression of the therapeutic protein and adenoviral proteins. A cytotoxic immune response is evoked against such transduced cells. It has been shown that cytotoxic T-lymphocytes (xe2x80x9cCTLsxe2x80x9d) directed against both the transgene product and products encoded by the vector are activated, following vector administration into immunocompetent animals (Song et al., Hum. Gene Ther. 8: 1207, 1997; Yang et al., J. Virol. 70: 7209, 1996). Activated CTLs subsequently eradicate transduced cells from the recipient. Consistent with this, the longevity of transgene expression is significantly extended in immuno-deficient and immuno-compromised animals.
Expression of at least some adenoviral genes in a target cell is at least in part due to background replication of the recombinant adenoviral vector genome and/or background activity of promoters driving the respective adenoviral genes (Yang et al., Nature Genet. 7: 362, 1994; Lusky et al., J. Virol. 72: 2022, 1998). As a result of the expression of at least some adenovirus proteins in a target cell in a recipient, an immune response may be mounted against transduced cells. Such an immune response is often not desired, especially when long-term expression of a transgene is aimed for. One mechanism by which adenovirus proteins in a target cell in a recipient may cause the immune system of the recipient to remove the target cell is the following. Proteins encoded by expressed adenovirus genes can be processed into small peptides in a proteosome of the target cell. Peptides produced during this processing can subsequently be presented at the cell surface of the transduced cells in the complex of MHC class-I and xcex2b2-microglobulin molecules. Finally, one or more of the peptides may be recognized as non-self peptides by circulating CTLs whereupon transduced cells can be eradicated from the recipient (reviewed in Ploegh, Science 280: 248, 1998).
In one aspect the present invention provides at least in part a solution to the problem of undesired removal of target cells in a recipient.
The present invention also provides, at least in part, a solution for the immune response against viral proteins. To this end, the invention provides improved recombinant adenoviral vectors that, in addition to deletion of E1, are also deleted for the adenoviral early 2A gene (xe2x80x9cE2A genexe2x80x9d or xe2x80x9cE2Axe2x80x9d). The protein encoded by E2A is expressed from recombinant E1-deleted adenoviral vectors. In addition to that, residual expression of E2A from E1-deleted recombinant adenoviral vectors induces the expression of the viral late genes, since DNA binding protein (xe2x80x9cDBPxe2x80x9d) has a positive regulatory effect on the adenovirus major late promoter (xe2x80x9cMLPxe2x80x9d) and, therefore, on the expression of the late genes (Chang et al., J. Virol. 64: 2103, 1990). Deletion of the E2A gene from the recombinant adenoviral genome will therefore improve the characteristics of recombinant adenoviral vectors. First, deletion of E2A will eliminate the synthesis of DBP. Second, it will inhibit the background replication of the recombinant adenoviral backbone. Third, it will reduce the residual expression of the late genes. Finally, it will increase the capacity of the vector to harbor larger and/or multiple transgenes.
The E2A gene encodes the 72-kDa protein single stranded DBP whose activity is pivotal for the adenovirus DNA replication (reviewed in The Molecular Repertoire of Adenoviruses II, Springer-Verlag 1995). Therefore, manufacturing of vectors that are deleted for E2A requires a cell line that complements for the deletion of E2A in the recombinant adenoviral vector. Major hurdles in this approach are:
a) that E2A should be expressed to very high levels and
b) that constitutive expression of E2A is toxic for cells and, therefore, impossible to achieve (Kiessig et al., Mol. Cell Biol. 4: 1354, 1984).
The current invention, therefore, involves the use of a temperature sensitive mutant of E2A derived from a temperature sensitive adenovirus under control of strong viral enhancer sequences, e.g., the cytomegalovirus enhancer for the generation of E2A complementing cell lines. DBP (ts125E2A) from hAd5ts125 is inactive at 39xc2x0 C., but is fully active at 32xc2x0 C. High levels of this protein can be maintained in the new complementing cells of the invention at the non-permissive temperature, until the switch is made to the permissive temperature. The invention also provides means and methods to use the complementing cell line, comprising E2A, tsE2A, or both E1 and tsE2A, for the generation of E2A- or E1- and E2A-deleted adenoviral vectors. The invention also involves inducible expression of E2A or tsE2A.
The invention also provides new cell lines that complement for E2A or for both the E1 and the E2A deletion in the vector. The invention also provides new recombinant adenoviral vectors deleted for E2A or both E1 and E2A.